Methods are known for detecting specific nucleic acids or analytes in a sample with high specificity and sensitivity. Such methods generally require first amplifying nucleic acid sequence based on the presence of a specific target sequence or analyte. Following amplification, the amplified sequences are detected and quantified. Conventional detection systems for nucleic acids include detection of fluorescent labels, colored dyes, fluorescent enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels.
One disadvantage of detection methods presently widely in use is the need to separate labeled starting materials from a final labeled product or by-product. Such separations generally require gel electrophoresis or immobilization of a target sequence onto a membrane for detection. Moreover, there are often numerous reagents and/or incubation steps required for detection.
It has been known that DNA and RNA polymerases are able to recognize and utilize nucleosides with a modification at or in place of the gamma position of the triphosphate moiety. It is further known that the ability of various polymerases to recognize and utilize gamma-modified nucleotide triphosphates (NTP's) appears to vary depending on the moiety attached to the gamma phosphate. In general, RNA polymerases are more promiscuous than DNA polymerases. Still, the efficiency of incorporation is significantly reduced compared to normal nucleotides. Even with this limitation, a number of potential applications using γ-labeled nucleoside triphosphates have been described in the literature.
A colorimetric assay for monitoring RNA synthesis from RNA polymerases in presence of a gamma-phosphate modified nucleotide has been previously reported (Vassiliou W et. al., Exploiting polymerase promiscuity: A simple calorimetric RNA polymerase assay, Virology. 2000 Sep. 1; 274(2):429–37; C. C. Kao et. al, U.S. Pat. No. 6,399,335 B1). In this prior report, RNA polymerase reactions were performed in the presence of a gamma-modified, alkaline phosphatase resistant nucleotide triphosphate which was modified at its gamma-phosphate with a dinitrophenyl group. When RNA polymerase reactions were performed in the presence of this gamma-modified NTP as the sole nucleotide triphosphate and a homopolymeric template, it was found that RNA polymerase could recognize and utilize the modified NTP. Moreover, when the polymerase reactions were performed in the presence of an alkaline phosphatase, which digested the p-nitrophenyl pyrophosphate aldo-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence was reported.
A number of references in the patent literature describe the use of γ-labeled nucleotides for DNA detection and sequencing (Hardin et. al., WO02/44425 A2, Williams, J. G. WO 00/36151 and WO 00/36152). Williams describes their use in single molecule detection of fluorescently labeled pyrophosphate being released after incorporation by polymerases. Attachment of a quencher to the base moiety allows for a homogeneous polymerase extension reaction where the amount of fluorescence in sample increases with incorporation of gamma labeled nucleoside triphosphate. Hardin et. al. further show their use in nucleic acid synthesis with a number of different polymerases. The efficiency of incorporation varies with polymerase used. Other reports (Felicia et. al., Arch. Biochem Biophys, 1986, 246, 564–571) describe the use of an γ-1,5-EDANS-ATP derivative as a substrate for E.Coli RNA polymerase. These references clearly point out the great potential that exists for the use of terminal phosphate labeled nucleotides. Unfortunately, despite the known potential uses, they have not been utilized in any major commercial applications.
As mentioned above, a major disadvantage with the use of γ-labeled nucleoside triphosphates in sequencing, SNP analysis and other assays is their poor acceptabilty by polymerases and other NTP utilizing enzymes. The reasons for this are probably multifold and may include the steric interactions between the gamma modification and certain amino acid residues in the enzyme pocket, and reduced metal binding by nucleotide or reduced electrostatic interactions between the nucleotide and polymerase due to one less negative charge on the nucleotide. It would, therefore, be of benefit to provide terminal labeled nucleoside polyphosphates where the label is further removed from the nucleoside by addition of additional phosphate groups, which also provide additional charges for metal binding or electrostatic interactions.
Nucleoside polyphosphates having a terminal modification and more than three phosphates are known in the literature. These are mainly dinucleoside polyphosphates (WO 01/12644 A1, U.S. Ser. No. 05/681,823, U.S. Ser. No. 05/663,322, U.S. Ser. No. 05/049,550, U.S. Ser. No. 05/658,890, U.S. Ser. No. 05/306,629, U.S. Ser. No. 04/886,749 and U.S. Ser. No. 6/183,978) where the modification on the terminal phosphate is the addition of another nucleoside. None of these have a label on the terminal phosphate. The only example of a nucleoside polyphosphate with a moiety on the terminal phosphate designed for detection and having four phosphate units that inventors are aware of is 5-bromo-4-chloro-3-indolyl tetraphospho-5′-adenosine. This compound has been used as a chromogenic substrate to investigate the activity of Ap4A phosphorylase and Ap4A hydrolases. In this case, detection was only possible after the tetraphosphate cleavage products were dephosphorylated and the indole moiety was oxidized in presence of nitro blue tetrazolium to give a colored dimer. This process requires at least two molecules of 5-bromo-4-chloro-3-hydroxyindole to generate a signal. At very low concentrations and especially for single molecule detection, this moiety is not useful. Thus, there is a need for terminal phosphate labeled nucleoside polyphosphates with readily detectable labels and which are better substrates for nucleic acid polymerases.
It would further be of benefit to provide nucleoside polyphosphates that are substrates for polymerases where the label on the terminal-phosphate could be varied so as to allow for chemiluminescent and fluorescent detection, analysis by mass or reduction potential, as well as for improved calorimetric detection, wherein only routine methods and instrumentation would be required for detection.
Given that DNA polymerases are known in the art to be less promiscuous than RNA polymerases regarding recognition and utilization of terminally-modified nucleotides, wherein the identity of the moiety at the terminal position can largely affect the DNA polymerase's specificity toward the nucleotide, it would be highly desired to provide for a non-radioactive method for detecting DNA by monitoring DNA polymerase activity. Furthermore, it would be desired that the synthesis and detection of DNA could be accomplished in a single-tube assay for real-time monitoring and that the label at the terminal-phosphate of nucleotide substrates could encompass chemiluminescent, fluorescent, and calorimetric detection, as well as analysis by mass or reduction potential.